HIV-1 infection depends on a series of molecular events involving biochemical interactions between viral components and host factors, resulting in fusion of viral and host membranes, reverse transcription, uncoating, nuclear entry, and integration. A key knowledge gap in the early stages of infection is the role of the viral capsid i facilitating reverse transcription. Mutations in the CA protein that destabilize the viral capsid result in impaired reverse transcription, indicating that the integrity of the viral capsid is critcal for efficient viral DNA synthesis. HIV-1 reverse transcriptase is a low processivity enzyme and dissociates from the template during strand transfer steps of reverse transcription. Employing a biochemical approach involving purified HIV-1 cores, we will test the hypothesis that the viral capsid serves as a vessel to maintain the local concentration of RT during reverse transcription and will study the role of restrictive host factors in HIV-1 capsid recognition and uncoating. We will also identify novel host factors that bind the HIV-1 capsid. The work will be organized by the following Specific Aims: Aim 1. To determine the mechanism by which the viral capsid facilitates HIV-1 reverse transcription. Aim 2. Identification of intersubunit interfaces critical for HIV-1 uncoating. Aim 3. To determine the effect of CPSF6-358 on HIV-1 uncoating. Aim 4. To determine the stoichiometric requirement for functional capsid recognition by TRIM5 restriction factors. Aim 5. Identification of novel capsid-interacting host factors. These studies will answer fundamental question in retrovirus biology and will clarify the mechanisms of restriction by endogenous host cell factors.